DNA sequencing is an important analytical technique of molecular biology. The development of sequencing techniques has led to advances in both the analysis and manipulation of genetic material.
Well-known methods of DNA sequencing include the Maxam-Gilbert chemical degradation method, described in Maxam et al., Meth. in Enzym. 65:499 (1980), and the Sanger dideoxy chain termination technique, described in Sanger et al., P.N.A.S. USA 74:5463 (1977). In each method DNA fragments labeled with .sup.32 P are generated which are analyzed by gel electrophoresis. Both methods are useful, although they can prove to be difficult and slow.
As a result, other methods have been sought, including those which do not rely upon short-lived radioisotopes, such as .sup.32 P. Several alternative methods of detection have been developed based on fluorescent labels. DNA fragments are labeled with one or more fluorescent dyes. Excitation with an appropriate light source (laser) causes a characteristic emission from the dye, thereby identifying the band. Even minute amounts of a biomolecule can be detected using such a method.
Among the fluorescent dyes which have been developed are a number of cyanine dyes which have been used to label various biomolecules for highly sensitive detection schemes. For example, U.S. Pat. No. 5,268,486, issued to Waggoner et al. (1993), discloses and claims fluorescent arylsulfonated cyanine dyes having large extinction coefficients and quantum yields for the purpose of detection and quantification of labeled components. Although the dyes described in this patent are useful, additional cyanine dyes are sought.
Recent advances in solid state laser technology have led to the commercial availability of inexpensive, reliable lasers with wavelengths near 680 nm. Suitable dyes excited with such lasers will fluoresce in the near infrared (NIR) region of the electromagnetic spectrum. These fluorescence signals will be free of background fluorescence from most biological systems. Unfortunately, there are few dyes available commercially which have suitable absorption/fluorescence properties and useful linking groups for attachment to biomolecules, and those that presently are available are quite expensive.
Accordingly, it is an object of the present invention to provide new cyanine dyes useful in labeling biomolecules.